Lyme disease is a multisystem illness caused by the tick-transmitted spirochete Borrelia burgdorferi (hereinafter referred to as "B. burgdorferi") (Burgdorfer, et al. 1982. Science 216:1317-1319; Steere, et al. 1983. N Engl J Med 308:733-740). Lyme borreliosis is the most common arthropod-borne infection in the United States and has been reported in many countries throughout Asia and Europe (Steere 1989. N Engl J Med 1:586-596). The early feature of the disease is a local infection of the skin, which may be followed by the development of systemic disease involving the nervous system, heart and joints (Steere 1989. N Engl J Med 1:586-596).
Culture of the spirochete from human body fluids and antigen detection methods often are falsely negative in the diagnosis of Lyme disease (Steere, et al. 1983. N Engl J Med 308:733-740; Benach, et al. 1983 N Engl J Med 308:740-742), leaving serological methods for antibodies to B. burgdorferi as the most appropriate currently available means for diagnosis. Most current diagnostic assays for Lyme disease utilize whole or sonicated B. burgdorferi cells as the test antigen, although many investigators have demonstrated improved performance of these tests when subcellular fractions of the spirochete were used (Grodzicki, et al. 1988. J Infect Dis 157:790-797; Magnareli, et al. 1989. J Infect Dis 159:43-49; Karlsson, et al. 1990. Eur J Clin Microbiol Infect Dis 9:169-177).
The flagellar protein is an immunodominant protein that generally elicits the earliest immune response after infection (Craft, et al. 1986. Clin Invest 78:934-939; Dattwyler, et al. 1989. Rev Infect Dis 11:1494-1498). Flagellin-enriched fractions of B. burgdorferi have been shown to improve the performance of Lyme diagnostic assays (Hansen, et al. 1988. J Clin Microbiol 26:338-346). The specificity of these assays, however, may be reduced because of cross-reactivity of B. burgdorferi flagellum with the flagella of other spirochetes, most notably with Treponema pallidum (hereinafter referred to as "T. pallidum"), the causative agent of syphilis (Magnarelli, et al. 1987. J Infect Dis 156:183-188). Current Lyme disease immunoassays utilize solubilized B. burgdorferi as the source of antigen, leading to false positive reactions from individuals with certain conditions, including syphilis, leptospirosis and other spirochetal infections. The lack of specificity is due to the fact that these organisms express similar antigens, especially the highly conserved flagellin protein. Thus, most Lyme disease immunoassays suffer from false positive reactions when syphilis positive patients are analyzed. Many institutions determine syphilis serologic status on all Lyme positive patients; if they are positive for syphilis they are considered to be negative for Lyme disease. This cross-reactivity with syphilis patients can be reduced by adsorption of the patient sera with the Reiter strain of Treponema (Magnarelli, et al. 1990. J Clin Microbiol 28:1276-1279), but this decreases the sensitivity of Lyme diagnostic assays.
The nucleotide and amino acid sequences have been determined for the flagellin protein of several B. burgdorferi isolates (Gassmann, et al. 1989. Nucleic Acids Res 17:3590; Wallich, et al. 1990. Infect Immun 58:1711-1719; Gassmann, et al. 1991 J Bacteriol 173:1452-1459; Collins, et al. 1991. Infect Immun 59:514-520). The entire flagellin protein contains 336 amino acids. Comparison of the conserved sequences with that of the T. pallidum endoflagellar protein (Pallesen, et al. 1989. Infect Immun 57:2166-2172) indicated high sequence homology at each end of the protein, but more variability in the central region. Collins, et al demonstrated that antibodies in the sera of Lyme and arthritis patients bound exclusively at the common amino-terminal region of the flagellin protein.
Wallich, et al., supra, merely speculated that the center region may be specific, based on comparison of amino acid sequences from similar organisms. Gassman, et al., (J. Bacteriol. 1991. 173:1452-1459) synthesized a series of overlapping octapeptides representing the entire sequence of the flagellum and analyzed serum from animals immunized with a variety of closely related bacteria. They demonstrated that the middle region from amino acid 180 to 260 only bound B. burgdorferi serum. Neither group demonstrated specificity using human sera. Significantly, Collins et al, supra observed that most Lyme patient sera bound to the amino-terminus region and their results indicated that a specific assay using flagellin was not possible.